1. Field of the Invention
The present invention relates to a method for tissue culture in vitro, more particularly, a method that places tissue blocks gathered into the hollow cavity of a porous dissoluble chamber, and then, by providing nutrients, the tissue blocks are to grow three-dimensionally toward the surrounding pores of the porous scaffold, thus new tissue is to be proliferated.
2. Description of the Prior Art
When human tissue is destructed or organs are destructed beyond repair due to accidents, aging or diseases, patients are often forced to experience limb dysfunctions or life-threatening crises, situations that may further lead to tremendous burden and loss for families and societies. Therefore, researchers indefatigably endeavor to seek appropriate tissue or organs for repair and replacement. In recent years, with the advancement of biotechnology, the biomedical material and culture technique for tissue cells are already combined to form a new field of research called tissue engineering. It is to be expected in the near future that, through the regeneration technology in the field of tissue engineering, the damaged tissue or organs can be repaired in vitro, or brand new tissue or organs can be produced in vitro to replace the damaged portions, thus the health of patients is recovered, and lives prolonged. Based upon the contents in the tissue engineering technology, a small portions of healthy tissue, either from patients or donors, are needed, so that tissue cells can be cultured profusely in vitro, and seeded into a dissoluble porous scaffolds; thus, with the three-dimensional framework of said scaffolds, the tissue cells are to adhere thereto and grow thereon. Later on the tissue cells in time grow and form three-dimensional tissue blocks, which then are implanted back to the areas needed repair. In accordance with histology, a block of tissue contains specific compositive cells, and the three-dimensional extracellular matrix (ECM) is to cover the areas between cells. The ECM not only sustains the framework of cells, but also manifests the specific functions of such tissue. Take the cartilage tissue culture for example, for the three-dimensional culture of the cartilage tissue in vitro, it is proven that the three-dimensional culture of the cartilage tissue with the scaffold being agarose gel can sustain the original cell forms and functions in tissue and the mutated and transformed cartilage cells cultured two-dimensionally are to be recovered to the original cartilage cell forms under the three-dimensional culture with the scaffold being agarose gel. For simulating the framework of ECM, various materials are developed, e.g., collagen or poly (glycolic co-Lactic) acid (PLGA), as well as various structures, e.g., fiber-mesh or porous artificial scaffolds. However, the most serious drawback for the aforementioned technology is that, when tissue cells are cultured in large quantity in the two-dimensional culture dish, the phenomenon of dedifferentiation, due to the process of culturing the tissue with three-dimensional alignment in a two-dimensional space, is to occur on those proliferated cells, thus the original forms and functions of cells are to be lost.
Furthermore, the seeding of cells is also a problem difficult to overcome. The pore diameters on the porous scaffolds should be larger than the diameters of the cells to provide cells with enough space to be developed into tissue, thus when the mixture of cells and the culture medium is seeded into the porous scaffold, cells are to overflow out of the chamber for it is difficult to keep cells inside the scaffold. In order to solve the problem, there are two methods available: first is the static seeding method, with the characteristics described as follows. At first, the density of cells cultured is to be adjusted to that higher than 106 cells/ml, and then, taking advantage of the water-containing nature of the porous scaffold, seeding cells are to be contained in the scaffold. After cells have all adhered to the scaffold, a large quantity of culture medium is added to start the culture process. Such method has the merits of knowing exactly how many the quantities of cells being seeded into the scaffold are, and high-density cells can be seeded into the scaffold. Nevertheless, the distribution of cells is still to be influenced by gravitational force, causing disproportional distribution between the upper layer and the lower layer of cells. Moreover, to prevent cells from overflowing, the volume of the culture medium mixed with cells is to be considerably limited, therefore the adhesion effect and the surviving rate shall both be taken into consideration. Another method is the dynamic seeding method of cells, with the characteristics described as follows. The spinner flask is used for spinning the water flow and thus cells are to be brought from the culture medium to the interior of the scaffold, a method that can obtain better adhesion rate of cells and better distribution of cells than that of the static seeding method. However, the drawbacks of the dynamic seeding method are as follows. The number of cells needed is higher, with the exact number of cells adhered in the interior of the scaffold being difficult to ascertain; also cells are seeded from the periphery, thus the density of cells in the periphery shall still be higher than that of cells in the interior. As a result, because cells in the periphery are having higher growth rate, a layer of hindrance is formed to prevent cells in the interior from exchanging nutrients, thus causing apparent disproportional proliferation between cells in the interior and those in the periphery of the scaffold.